Soil extract materials, particularly the classes of substances known collectively as "humus," "humics," "humic acid(s)," or "humates," have been widely used in a number of applications for many years, as reviewed by F. J. Stevenson, Humus Chemistry. Genesis Composition Reactions; New York: Wiley, 1964; and, more recently, by A. Piccolo, Humic Substances in Terrestrial Ecosystems; New York: Elsevier, 1996.
Natural and synthetic soil extracts have already been used extensively in horticultural and related industries, particularly as soil enhancement as well as soil remediation agents. In addition, natural and synthetic soil extracts have been employed as additives in organic gardening and landscaping; and in fresh-water aquaria. Some medicinal benefits have also been claimed for both synthetic- and naturally-occurring soil extract substances.
R. H. Faust, in a paper presented at the Conference of the International Federation of Organic Agriculture Movements; Copenhagen, Denmark: October, 1996; P2, 20, has documented the benefits of humates in agriculture. In general, it has been found that humic materials can stimulate plant growth, including crop yield, by about 10-30%.
Soil extracts, and humic acid in particular, chelate a variety of metals. As a result, humic materials have been employed in soil remediation to remove heavy-metal contamination, as reported by M. A. Rashid, Soil Sci. 1971, 111, 298-306. Humic acid has also been used to enhance the removal of aromatic hydrocarbons from aquifers contaminated with petroleum products: H. Xu, S. Lesage, L. Durham, and K. Novakowski, in Proceedings of the Fourth Annual Symposium on Groundwater and Soil Remediation; Calgary Alberta: Sep. 21-23, 1994; 635-646; S. Lesage, H. Xu, K. S. Novakowski, S. Brown, and L. Durham, in Proceedings of the Fifth Annual Symposium on Groundwater and Soil Remediation; Toronto, Ontario: Oct. 2-6, 1995.
Humate materials have been used as poultry feed additives. Adding humate materials to the fodder of broiler chickens increases the yield mass on average by 5-7%, and also provides for a 3-5% gain in poultry safety: L. M. Stepchenko, L. V. Zhorina, and L. V. Kravtsova, Biol. Nauki 1991, 10, 90-95.
T. A. Huck, N. Porter, and M. E. Bushell, J. Gen. Microbiol. 1991, 137(10), 2321-2329, have reported that soil isolates are effective media additives for the production of antibiotics, and that the extent of microbial growth stimulation can be quite large depending upon the species, the culture medium, and the environment. The use of selected batches of soil lignite humate as culture media for isolating thermophilic Campylobacter species extracts has also been documented by K. Weinrich, K. Winkler, and E. Heberer, DTW Dtsch. Tierarztl Wochenschr. 1990, 97(12), 511-515. In addition, B. Grunda, Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. 1970, 125(6), 584-593, has described the effects of humic acid on the count of soil microorganisms in culture.
Humates have long been used as folk remedies for a wide variety of illnesses (F. K. Achard, Crells Chem. Ann. 1786, 11, 391-403), as recounted by T. D. Lotosh, Biol. Nauki 1991, 10, 99-103.
Humic acids isolated from peat exhibited significant efficacy for adhesions when tested on female rats that had standardized lesions placed on both uterine horns and the peritoneum of the anterior abdominal wall: M. Mesrogli, D. H. Maas, B. Mauss, S. Plogmann, W. Ziechmann, and J. Schneider, Zentralbl. Gynakol. 1991, 113(10), 583-590.
The ability of natural humic acid to affect anaphylactic sensitization and mast cell secretory function has been established by J. Wyczolkowska, T. Michon, Z. Slusarczyk, B. Kolago, and C. Maslinski, Acta Pol. Pharm. 1993, 50(6), 475-480. Humic substances in doses of 20 and 50 milligrams per kilogram body weight reduced histamine release from mouse peritoneal mast cells challenged with anti-IgE or concanavalin A in vitro.
Humic substances, including peats and sodium humates, are known to exhibit anti-inflammatory properties: M. Kuhnert, V. Fuchs, and S. Golbs, Arch. Exp. Veterinarmed. 1982, 36(2), 169-177; S. B. Ye, J. Y. Chen, and Z. X. Zeng, Ssu Chuan I Hsueh Yuan Hsueh Pao 1985, 16(2), 127-129. Inflammatory states of the cervix, especially cervical erosion (known generally as cervicitis), can be treated with humic preparations: J. Woyton, M. Gabrys, T. Bielanow, M. Zimmer, J. Sokalski, R. Geneja, and M. Zborowski, Arch. Immunol. Ther. Exp. (Warsz) 1993, 41(1), 99-103.
Humic substances have been known to exhibit anti-microbial properties. Species for which natural as well as synthetic humic substances have been shown to be inhibitory include C. albicans, Ent. cloacae, Prot. vulgaris, Ps. aeruginosa, S. typhimurium, St. aureus, St. epidermidis, Str. pyogenes (R. Ansorg and W. Rochus, Arzneimittelforschung 1978, 28(12), 2195-2198; E. coli and Str. faecalis were not affected), and Str. mutans (sobrinus) (Y. Nakamura, H. Kuwashima, S. Aoki, and T. Masuhara, Shika Kiso Igakkai Zasshi 1989, 31(3), 329-332). Broadly speaking, concentrations in the range 50-2000 parts per million (ppm) are usually effective, yet are not cytotoxic: K. D. Thiel, B. Helbig, R. Klocking, P. Wutzler, M. Sprossig, and H. Schweizer, Pharmazie 1981, 36(1), 50-53.
Humic substances have long been known to exhibit anti-viral properties (H. Schultz, Dtsch. Tierarztl. Wochenschr. 1962, 69, 613; 1965, 72(13), 294-297; R. Klocking and M. Sprossig, Experientia 1972, 28(5), 607-608), particularly retroviruses (G. Sydow, V. Wunderlich, R. Klocking, and B. Helbig, Pharmazie 1986, 41(12), 865-868). Viral pathogens for which soil-extract materials have been shown to be effective include in particular Coxsackie virus A9 (Griggs-Baylor) (R. Klocking and M. Sprossig, Experientia 1972, 28(5), 607-608), herpes simplex virus type 1 (B. T. Rouse (Ed.), Herpes Simplex Virus; Berlin: Springer-Verlag, 1992; R. Klocking, K. D. Thiel, P. Wutzler, B. Helbig, and P. Drabke, Pharmazie 1978, 33(8), 539; F. Schiller, R. Klocking, P. Wutzler, and I. Farber, Dermatol. Monatsschr. 1979, 165(7), 505-509; B. Helbig, A. Sauerbrei, R. Klocking, P. Wutzler, N. Wicht, U. Wiedemann, and G. Herrmann, J. Med. Virol. 1987, 23(3), 303-309; R. Klocking and B. Helbig, in Humic Substances in the Aquatic and Terrestrial Environment; Berlin: Springer-Verlag, 1991; 407-412;) and type 2 (anon. Zentralbl. Bakteriol Orig. A! 1976, 234(2), 159-169; K. D. Thiel, R. Klocking, H. Schweizer, and M. Sprossig, Zentralbl. Bakteriol Orig. A! 1977, 239(3), 304-321; K. D. Thiel, B. Helbig, R. Klocking, P. Wutzler, M. Sprossig, and H. Schweizer, Pharmazie 1981, 36(1), 50-53; K. D. Thiel, B. Helbig, M. Sprossig, R. Klocking, and P. Wutzler, Acta Virol. 1983, 27(3), 200-208; K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782); human immunodeficiency virus (HIV) (M. Cushman, P. Wang, S. H. Chang, C. Wild, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 329-337; M. Cushman, S. Kanamathareddy, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 337-342; D. Schols, P. Wutzler, R. Klocking, B. Helbig, and E. De Clercq, J. Acquir. Immune Defic. Syndr. 1991, 4(7), 677-685; S. Loya, R. Tal, A. Hizi, S. Issacs, Y. Kashman, and Y. Loya, J. Nat. Prod. 1993, 56(12), 2120-2125; J. Schneider, R. Weis, C. Manner, B, Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395; influenza virus type A (Krasnodar/101/59/H2N2) (R. Mentel, B. Helbig, R. Klocking, L. Dohner, and M. Sprossig, Biomed. Biochim. Acta 1983, 42(10), 1353-1356); and type B (J. Hils, A. May, M. Sperber, R. Klocking, B. Helbig, and M. Sprossig, Biomed. Biochim. Acta 1986, 45(9), 1173-1179); as well as other respiratory tract infectious agents (A. Jankowski, B. Nienartowicz, B. Polanska, and A. Lewandowicz-Uszynska, Arch. Immunol. Ther. Exp. (Warsz) 1993, 41(1), 95-97).
The mechanism whereby humic substances inhibit the cytopathicity of a number of viruses has been studied in some detail. It is thought that the materials prevent viral replicating by sorbing onto the viral envelope protein (gp120SU in the case of HIV) and thereby blocking the sorption of viral particles to cell surfaces: K. D. Thiel, R. Klocking, H. Schweizer, and M. Sprossig, Zentralbl. Bakteriol. Orig. A! 1977, 239(3), 304-321; D. Schols, P. Wutzler, R. Klocking, B. Helbig, and E. De Clercq, J. Acquir. Immune Def. Syndr. 1991, 4(7), 677-685; anon., Fortschr. Med. 1995, 113(7), 10; J. Schneider, R. Weis, C. Manner, B. Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395. Extracellular interception of pathogens by chemical agents that bind to them is a well-known means of immunological defense (D. M. Shankel, S. Kuo, C. Haines, and L. A. Mitscher, in Antimutagenesis and Anticarcinogenesis Mechanisms III; G. Bronzetti, H. Hayatsu, S. De Flora, M. D. Waters, and D. M. Shankel (Eds.); New York: Plenum, 1993; 65-74). Such materials might well be termed "despathogens," following the terminology proposed by T. Kada and K. Shimoi, Bioessays 1987, 7, 113-116, regarding "desmutagens."
It has been reported that heat treatment of humic acids at 120 degrees Centigrade for 15 minutes does not alter their inhibitory effect on mutagens: T. Sato, Y. Ose, and H. Nagase, Mutat. Res. 1986, 162(2), 173-178; T. Sato, Y. Ose, H. Nagase, and K. Hayase, Sci. Total Environ. 1987, 62(4), 305-310). That is, humic acids can be sterilized by autoclaving.
A direct comparison of enzymatic- with nonenzymatic-synthesized humic acid has shown that the latter is about a factor of ten more effective than the former for the treatment of herpes types 1 and 2: K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782.
Implanted bovine calcium hydroxyapatite is highly osteoconductive, and serves the host tissue as a "guideline" for the deposition of newly developing bone tissue. However, while it is well tolerated, it is resorbed only very slowly. Impregnation of the bovine hydroxyapatite with synthetic humic acid measurably stimulates the resorption process.
There is extensive covalent as well as hydrogen bonding of humic substances to collagen fibers (with undoubted crosslinking as well), as determined by x-ray diffraction analysis: U. N. Riede, I. Jonas, B. Kirn, U. H. Usener, W. Kreutz, and W. Schlickewey, Arch. Orthop. Trauma Surg. 1992, 111(5), 259-264. Tendon strength is thereby increased by as much as 75 percent.
Natural as well as synthetic humic acids have been found to stimulate the phagocytic and bactericidal activity of granulocytes in humans at dose levels of 100-300 milligrams per day over a 14-day testing period: U. N. Riede, G. Zeck-Kapp, N. Freudenberg, H. U. Keller, and B. Seubert, Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1991, 60(1), 27-34; M. Kowalska, A. Denys, and J. Bialek, Acta Pol. Pharm. 1993, 50(4-5), 393-395. Of additional interest is the finding that dose levels of 600 milligrams per day caused only a transient and insignificant increase of phagocytic and bactericidal properties of the granulocytes.
The influence of natural as well as synthetic humic acids on haemostasis has been studied: H. P. Klocking, Arch. Toxicol. Suppl. 1991, 14, 166-169; W. Buczko, B. Malinowska, M. H. Pietraszek, D. Pawlak, and E. Chabielska, Acta Pol. Pharm. 1993, 50(6), 507-511. It was found that humic acid in dose levels of 100-300 milligrams per kilogram body weight had no effect on bleeding time, clotting time, thrombin time, prothrombin time, kaolin-kephalin time, euglobulin lysis time, the concentration of fibrinogen, the platelet count, or ADP-induced platelet aggregation.
Various synthetic humic acids have been found to inhibit strongly the activity of purified lipoxygenase of rabbit reticulocytes, whereas prostaglandin H synthase of sheep vesicular gland is only weakly inhibited: C. Schewe, R. Klocking, B. Helbig, and T. Schewe, Biomed. Biochim. Acta 1991, 50(3), 299-305. The most effective humic acids were those derived from caffeic acid, 2,5-dihydroxytoluene, and 3,4-dihydroxytoluene.
The effect of natural humic acid on the regenerative response of liver tissue has been examined in rats submitted to two-thirds hepatectomy. The results were thought to be two-fold in nature. First, the short-term application of humic acid at a dose of 20 milligrams per kilogram body weight per day inhibited ornithine decarboxylase activity, as well as caused a decrease in spermidine formation and DNA and RNA, resulting in an overall decrease in liver restitution. In contrast, long-term application of humic acid resulted in the stimulation of ornithine decarboxylase, an increase in spermidine and histamine as well as RNA and DNA levels, and in overall liver mass. The effects might be due at least in part to the humic-acid inhibition of polyamine biosynthesis: C. Maslinksi, W. A. Fogel, and W. Andrzejewski, Acta Pol. Pharm. 1993, 50(4-5), 413-416.
Humic as well as fulvic acids extracted from peat have been shown to stimulate respiration in rat liver mitochondria when present at concentrations of 40-360 micrograms per milliliter. Humic substances at concentrations of 40-400 micrograms per milliliter also increased the efficiency of oxidative phosphorylation in mitochondria in vitro, particularly after contact periods of over 1 hour: S. A. Visser, Sci. Total Environ. 1987, 62(4), 347-354.
Natural, synthetic, and commercial humic acids all have the ability to inhibit human plasmin activity: F. J. Lu and Y. S. Lee, Sci. Total Environ. 1992, 114(4), 135-139. Thus, at a concentration of 20 micrograms per milliliter, each resulted respectively in residual plasmin activities of 70, 93, and 40 percent. Synthetic humic acids fabricated from caffeic acid and 3,4-dihydroxyphenylacetic acid have also been found to raise the activity of plasminogen activator in isolated vascular preparations of pig ear (H. P. Klocking, R. Klocking, and B. Helbig, Farmakol. Toksikol. 1984, 47(1), 93-95).
Peat-derived natural humic acids have been found to inhibit the hydrolysis of N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-leucine methyl ester by alpha-chymotrypsin as well as by subtilisin: Sh. Zh. Zhorobekova and K. A. Kydralieva, Biol. Nauki 1991, 10, 151-154.
Sodium humate has been found to increase the lifespan of mongrel rats exposed to lethal doses of .sup.60 Co-radiation, as reported by G. G. Pukhova, N. A. Druzhina, L. M. Stepchenko, and E. E. Chebotarev, Radiobiologiia 1987, 27(5), 650-653.
It has been found that naturally-occurring humic acid preparations can stimulate the production of cytokines, including interferon-gamma, interferon-alpha, and tumor necrosis factor-alpha (A. D. Inglot, J. Zielinksa-Jenczylik, and E. Piasecki, Arch. Immunol. Ther. Exp. (Warsz) 1993, 41(1), 73-80); and interferon-beta (Z. Blach-Olszewska, E. Zaczynksa, E. Broniarek, and A. D. Inglot, Arch. Immunol. Ther. Exp. (Warsz), 1993, 41(1), 81-85).
Histopathological and ultrastructural studies have shown that naturally-occurring humic acids can cause morphological changes characteristic of thymus activity stimulation: J. A. Madej, J. Kuryszko, and T. Garbulinski, Acta Pol. Pharm. 1993, 50(4-5), 397-404.
It has been shown that incubation of cultured human umbilical vein endothelial cells either with natural or synthetic humic acid results in an enhanced cell surface expression of tissue factor activity. There are also changes in intracellular divalent calcium levels: H. L. Yang, F. J. Lu, S. L. Wung, and H. C. Chiu, Thromb. Haemost. 1994, 71(3), 325-330.
Natural humic acid administered prophylactically to rats can decrease significantly the amount of gastric mucosa damage induced with ethanol. Humic acid also significantly accelerates the healing process of experimental-induced gastric and duodenal ulcers: T. Brzozowski, A. Dembinski, and S. Konturek, Acta Pol. Pharm. 1994, 51(1), 103-107.
Humic acids have also been employed as veterinary medicine therapies, as described and discussed by M. Kuhnert, V. Fuchs, H. Knauf, and U. Knoll, Arch. Exp. Veterinarmed. 1985, 39(3), 344-349; and by M. Kuhnert, V. Fuchs, and S. Golb, Dtsch. Tierarztl. Wochenschr. 1989, 96(1), 3-10. For example, H. Schultz, Dtsch. Tierarztl. Wochenschr. 1962, 69, 613; 1965, 72(13), 294-297, successfully employed peat mull to prevent the transmission of foot and mouth disease in pigs.
The pharmacokinetics of sodium humate in chickens have been studied extensively by J. Hampl, I. Herzig, and J. Vlcek, Vet. Med. (Praha), 1994, 39(6), 305-313. Free or liposome-encapsulated sodium humate was administered to chickens intracardially, orally, or subcutaneously and a number of pharmacokinetic parameters were then determined. The blood clearance of liposome-encapsulated sodium humate was higher than that of free sodium humate regardless of the manner of administration. On the other hand, the elimination half-life was longer after extravascular than after intracardial administration. Maximal drug concentration values indicated that the penetration of sodium humate from the injection site into blood circulation is very slow. Biological availability of sodium humate also depended on the method of administration and dosage form. Aside from intracardial administration, the highest bioavailability was found after subcutaneous administration of free sodium humate. Synthetic humic acid has been found to penetrate the dermis very quickly from a 1 percent water/oil emulsion, and to then form a reservoir in the horny layer: W. Wohlrab, B. Helbig, R. Klocking, and M. Sprossig, Pharmazie 1984, 39(8), 562-564. Also, about 30 minutes after external application, concentrations of 1-3 percent of the total quantity applied are achieved, which percentage remains essentially unchanged thereafter.
The toxicity of naturally-occurring humic acids is remarkably low (K. D. Thiel, B. Helbig, R. Klocking, P. Wutzler, M. Sprossig, and H. Schweizer, Pharmazie 1981, 36(1), 50-53; U. N. Riede, I. Jonas, B. Kirn, U. H. Usener, W. Kreutz, and W. Schlickewey, Arch. Orthop. Trauma Surg. 1992, 111(5), 259-264; H. Czyzewska-Szafran, Z. Jastrzebski, D. Soltysiak-Pawluczuk, M. Wutkiewicz, A. Jedrych, and M. Remiszewska, Acta Pol. Pharm. 1993, 50(4-5), 373-377; H. L. Yang, F. J. Lu, S. L. Wung, and H. C. Chiu, Thromb. Haemost. 1994, 71(3), 325-330). Cytotoxic effects of antiviral substances, including humic acids, are usually evaluated via biological (viability and alterations of cell morphology) and biochemical testing methods (.sup.51 Cr release), as described by K. D. Thiel, U. Eichhorn, H. Schweizer, and R. Klocking, Arch. Toxicol. Suppl. 1980, 4, 428-430.! The cytotoxicity (CD.sub.50) of a naturally-occurring humic acid for human peripheral blood leukocytes (PBL) was found to be 1-9 milligrams per milliliter. In addition, J. Schneider, R. Weis, C. Manner, B. Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395, reported that the cytotoxicity of a synthetic humic acid prepared from hydroquinone for MT-2 cells was approximately 600 micrograms per milliliter. It has also been found that medicaments prepared from humic acids isolated from naturally-occurring soil materials are neither carcinogenic (Syrian hamster embryo cell transformation test: J. Koziorowska and E. Anuszewska, Acta Pol. Pharm. 1994, 51(1), 101-102) nor mutagenic (T. Sato, Y. Ose, and H. Hagase, Mutat. Res. 1986, 162(2), 173-178; V. M. Sui, A. I. Kiung, and T. I. Veidebaum, Vopr. Kurortol. Fiozioter. Lech. Fiz. Kult. 1986, 2(3-4), 34-37; J. Koziorowska, B. Chlopkiewicz, and E. Anuszewska, Acta Pol. Pharm. 1993, 50(4-5), 379-382). Prenatal (S. Golbs, V. Fuchs, M. Kuhnert, and C. Polo, Arch. Exp. Veterinarmed. 1982, 36(2), 179-185) and embryotoxic and teratogenic effects (T. Juszkiewicz, M. Minta, B. Wlodarczyk, B. Biernacki, and J. Zmudzki, Acta Pol. Pharm. 1993, 50(4-5), 383-388) are also not observed with humic preparations at daily dose levels from 5-50 milligrams per kilogram body weight. Topical preparations are tolerated even better (V. V. Soldatov and M. N. Cherepanova, Vopr. Kurortol. Fizioter. Lech. Fiz. Kult. 1970, 35(3), 256-259; H. Czyzewska-Szafran, Z. Jastrzebski, D. Soltysiak-Pawluczuk, M. Wutkiewicz, A. Jedrych, and M. Remiszewska, Acta Pol. Pharm. 1993, 50(4-5), 373-377) when applied dermally in aqueous solution in amounts as high as 10 percent weight-by-volume (K. Wiegleb, N. Lange, and M. Kuhnert, Dtsch. Tierarztl. Wochenschr. 1993, 100(10), 412-416).
Soil extracts, including humics, are quite complex mixtures of organic and inorganic polymeric compounds whose composition varies widely depending upon the source of the soil and the method(s) of extraction and subsequent treatment: D. Vaughan and R. E. Malcolm, Plant Soil Sci. 1985, 16, 1-443 (see also N. Senesi, Y. Chen, and M. Schnitzer, Soil Biol. Biochem. 1977, 9, 397-403).
Techniques used for the chemical characterization of soil extracts, including humics, have included capillary electrophoresis (S. Pompe, K. Heise, and H. Nitsche, J. Chromatogr. A, 1996, A723(1), 215-218), ultracentrifugation (R. S. Cameron, B. K. Thornton, R. S. Swift, and A. M. Posner, J. Soil Sci. 1972, 23(4), 394-408; A. E. Wilkinson, J. J. Higgo, and M. N. Jones, Biochem. Soc. Trans. 1991, 19(4), 414S), electron paramagnetic resonance and infrared spectroscopy (G. Tollin and C. Steelink, Biochim. Biophys. Acta, 1966, 112(2), 377-379), various solvent and other fractionation methods (R. S. Cameron, B. K. Thornton, R. S. Swift, and A. M. Posner, J. Soil Sci. 1972, 23(4), 394-408; C. E. Clapp, M. H. Hayes, and R. S. Swift, Agricultural Research Service Report Number 0000042025; M. H. Hayes, R. L. Malcolm, and C. E. Clapp, Agricultural Research Service Report Number 0000042035; I. Csiky, G. Marko-Varga, and J. A. Jonsson, Anal Chim. Acta 1985, 178, 307-312; J. A. Amador, P. J. Milne, C. A. Moore, and R. G. Zika, Mar. Chem. 1990, 29, 1-17), gas chromatography (I. Arsenie, H. Boren, and B. Allard, Sci. Total Environ. 1992, 116(3), 213-220), gas chromatography-mass spectrometry (H. -R. Schulten and M. Schnitzer, Soil Sci. 1992, 153(3), 205-224; G. Chiavari, G. Torsi, D. Fabbri, and G. C. Galletti, Analyst (London) 1994, 119(6), 1141-1150), gel-permeation chromatography (B. Kosinkiewicz, Acta Microbiol. Pol. 1977, 26(4), 387-392; S. Mori, M. Hiraide, and A. Mizuike, Anal. Chim. Acta 1987, 193, 231-238), high-performance liquid chromatography (M. A. Curtis, A. F. Witt, S. B. Schram, and L. B. Rogers, Anal. Chem. 1981, 53, 1195-1199; K. Ravichandran, J. J. Lewis, I. -H. Yin, M. Koenigbauer, C. R. Powley, P. Shah, and L. B. Rogers, J. Chromatogr. 1988, 439, 213-226; J. Knuutinen, L. Virkki, P. Mannila, P. Mikkelson, J. Paasivirta, and S. Herve, Wat. Res. 1988, 22(8), 985-990, M. Susic and K. G. Boto, J. Chromatogr. 1989, 482(1), 175-187), mass spectrometry (H. -R. Schulten, G. Abbt-Braun, and F. H. Frimmel, Environ. Sci. Technol. 1987, 21(4), 349-357; C. Sorge, R. Mueller, P. Leinweber, and H. R. Schulten, Fresenius' J. Anal. Chem. 1993, 346(6-9), 697-703; M. Remmler, A. Georgi, and F. -D. Kopinke, Eur. Mass Spectrom. 1995, 1(4), 403-407), nuclear magnetic resonance (F. J. Vila, H. Lentz, and H. D. Ludemann, Biochem. Biophys. Res. Commun. 1976, 72(3), 1063-1070; G. Almendros, R. Frund, F. J. Gonzalez-Vila, K. M. Haider, H. Knicker, and H. D. Ludemann, FEBS Lett. 1991, 282(1), 119-121), and polyacrylamide gel electrophoresis (R. Klocking, J. Chromatogr. 1973, 78, 409-416; L. P. Glazkova, V. S. Ulashchik, and F. A. Puntus, Vopr. Kurortol. Fizioter. Lech. Fiz. Kult. 1984, 2(2), 21-24).
Very many studies have been carried out on the structural characterization of soil extracts, including humic acid, by reductive degradation, as reviewed by L. B. Sonnenberg, Ph.D. Thesis, University of North Carolina at Chapel Hill, 1989: Dissertation Services Order No. 9007318. Models of humic structure based on the physicochemical properties of membranes have also been developed by R. L. Wershaw, Environ. Health Perspect. 1989, 83(11), 191-203. R. R. Engebretson and R. von Wandruszka, Environ. Sci. Technol. 1994, 28, 1934, have described efforts at characterizing the micro-organization of dissolved humic acids in terms of their secondary structure, that is, on the way in which these large molecules arrange themselves in three dimensions in solution. The molecules are thought to be dendritic, that is, are hyperbranched fractal-like structures that emanate somewhat like the spokes of a wagon-wheel from a central core, and which contain a large number of carboxyl and hydroxyl terminal groups: T. H. Mourey, S. R. Turner, M. Rubinstein, J. M. J. Frechet, C. J. Hawker, and K. L. Wooley, Macromolecules 1992, 25, 2401-2406. Cluster aggregates of humic acid have an average diameter of 700-1700 Angstroms; large clusters have a fractal dimension of 2.3: R. Osterberg and K. Mortensen, Radiat. Environ. Biophys. 1994, 33(3), 269-276.
Because humic substances are not chemically well-defined, the preparation of synthetic humic acids whose physicochemical properties mimic naturally-occurring materials is quite difficult, as pointed out by K. Murray and P. W. Linder, J. Soil Sci. 1983, 34, 511-523. Nevertheless, there have been several notable advances in this area. Broadly speaking, three general strategies have evolved. All depend upon starting with well-defined molecules of molecular weight on the order of hydroxybenzoic acid, and then causing the molecules to polymerize upon themselves to form larger molecules. The methods differ in the causation factor, which can be microbial, chemical, or enzymatic.
Humic acids of microbial origin have been described and discussed by M. Robert-Gero, C. Hardisson, L. Le Borgne, and G. Pignaud, Ann. Inst. Pasteur (Paris) 1966, 111(6), 750-767; and by M. Robert-Gero, C. Hardisson, L. Le Borgne, and G. Vidal, Ann. Inst. Pasteur (Paris) 1967, 113(6), 903-909.
The chemical synthesis of humic acids has been pioneered by R. Klocking, B. Helbig, and associates: R. Klocking, B. Helbig, and P. Drabke, Pharmazie 1977, 32, 297; R. Klocking, B. Helbig, K. D. Thiel, T. Blumohr, P. Wutzler, M. Sprossig, and F. Schiller, Pharmazie 1979, 34(5-6), 293-294; R. Mentel, B. Helbig, R. Klocking, L. Dohner and M. Sprossig, Biomed. Biochim. Acta 1983, 42(10), 1353-1356; H. P. Klocking, R. Klocking, and B. Helbig, Farmakol. Toksikol. 1984, 47(1), 93-95; K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782; J. Hils, A. May, M. Sperber, R. Klocking, B. Helbig, and M. Sprossig, Biomed. Biochim. Acta 1986, 45(9), 1173-1179; B. Helbig, A. Sauerbrei, R. Klocking, P. Wutzler, N. Wicht, U. Wiedemann, and G. Herrmann, J. Med. Virol. 1987, 23(3), 303-309; K. I. Hanninen, R. Klocking, and B. Helbig, Sci. Total Environ. 1987, 62, 201-210; R. Klocking and B. Helbig, in Humic Substances in the Aquatic and Terrestrial Environment; New York: Springer-Verlag, 1989; 407-412; C. Schewe, R. Klocking, B. Helbig, and T. Schewe, Biomed. Biochim. Acta 1991, 50(3), 299-305; D. Schols, P. Wutzler, R. Klocking, B. Helbig, and E. De Clercq, J. Acquir. Immune Defic. Syndr. 1991, 4(7), 677-685. Typically, 10 millimoles of the starting small-molecule phenolic compound is dissolved in distilled water, the pH is adjusted to 8.5 with aqueous sodium hydroxide (NaOH), and then 2-5 millimoles of sodium periodate (NaIO.sub.4) is added. The solution is warmed at 50.degree. C. for 30 minutes, and is then allowed to stand overnight. The resultant humic acid-like polymeric products are isolated by precipitation with lead(II) nitrate Pb(NO.sub.3).sub.2 !. The precipitated polymers are redissolved in aqueous sodium hydroxide (pH 8.5) and heated with 8-hydroxyquinoline for 30 minutes at 100.degree. C. The precipitate formed is lead(II) chelate, which is removed by filtration. Residual 8-hydroxyquinoline is extracted with chloroform, and the desired polymeric material is then precipitated from the aqueous solution by the addition of various combinations of acetic acid, ethyl acetate, and ethanol. Starting compounds that have been used for the synthesis of humic-like materials include 4-bis(p-hydroxyphenyl)methylene!-2,5-cyclohexadie-1-one (aurin), 4-bis(3-carboxy-4-hydroxyphenyl)methylene!-2-carboxy-2,5-cyclohexadien-1- one (aurintricarboxylic acid), 3-(3,4-dihydroxyphenyl)propenoic acid (caffeic acid), 1,2-dihydroxybenzene (catechol), 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid 3-(3,4-dihydroxyphenyl)propenoate (chlorogenic acid), 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid), 1-(3,4-dihydroxyphenyl)-2-(N-methylamino)ethanol (epinephrine), 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid (ferulic acid), 3,4-5-trihydroxybenzoic acid (gallic acid), 2,5-dihydroxybenzoic acid (gentisic acid), 2,5-dihydroxyphenylacetic acid (homogentisic acid), 3-(3,4-dihydroxyphenyl)propionic acid (hydrocaffeic acid), 1,4-dihydroxybenzene (hydroquinone), 2,3-dihydroxytoluene (3-methylcatechol), 3,4-dihydroxytoluene (4-methylcatechol), 2,5-dihydroxytoluene (2-methylhydroquinone), 4,4'-(2,3-dimethyltetramethylene)-di-(1,2-dihydroxybenzene) (nordihydroguaiaretic acid), 1-(3,4-dihydroxyphenyl)-2-aminoethanol (norepinephrine), 3,4-dihydroxybenzoic acid (protocatechuic acid), 1,2,3-trihydroxybenzene (pyrogallol), 1,3-dihydroxybenzene (resorcinol), and 4-hydroxy-3-methoxybenzoic acid (vanillic acid). Other notable efforts on the chemical synthesis of humic-like substances include the studies by De Clercq and colleagues on aurintricarboxylic acid, its derivatives, and related compounds: M. Cushman, P. Wang, S. H. Chang, C. Wild, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 329-337; M. Cushman, S. Kanamathareddy, E. De Clercq, D. Schols, M. E. Goldman, and J. A. Bowen, J. Med. Chem. 1991, 34(1), 337-342. Related efforts have also been reported by M. Robert-Gero, C. Hardisson, L. Le Borgne, and G. Vidal, Ann. Inst. Pasteur (Paris) 1967, 113(6), 903-909; M. Jakubiec, E. Miszczak, and J. Szczerkowska, Acta Microbiol. Pol. B! 1971, 3(1), 63-66; R. Ansorg and W. Rochus, Arzneimittelforschung 1978, 28(12), 2195-2198; J. Pommery, M. Imbenotte, A. F. Urien, D. Marzin, and F. Erb, Mutat. Res. 1989, 223(2), 183-189; F. J. Lu and Y. S. Lee, Sci. Total Environ. 1992, 114, 135-139; K. Wiegleb, N. Lange, and M. Kuhnert, DTW Dtsch. Tierarztl. Wochenschr. 1993, 100(10), 412-416; H. L. Yang, F. J. Lu, S. L. Wung, and H. C. Chiu, Thromb. Haemost. 1994, 71(3), 325-330; W. Seffner, F. Schiller, R. Heinze, and R. Breng, Exp. Toxicol. Pathol. 1995, 47(1), 63-70; and J. Schneider, R. Weis, C. Manner, B. Kary, A. Werner, B. J. Seubert, and U. N. Riede, Virology 1996, 218(2), 389-395.
The enzymatic catalytic synthesis of humic acids dates to about 1961 with the work by R. E. Hampton and R. W. Fulton, Virology 1961, 13, 44-52 (see also R. E. Hampton, Phytophathology 1970, 60, 1677-1681), who found that enzymatically oxidized phenols inactivate phytopathogenic (i.e., plant-related) viruses. Typically o-diphenol oxidase has been employed for the enzymatic synthesis of humic-like materials: anon. Zentralbl. Bakteriol. Orig. A! 1976, 234(2), 159-169; R. Klocking, B. Helbig, and P. Drabke, Pharmazie 1977, 32(5), 297; K. D. Thiel, B. Helbig, R. Klocking, P. Wutzler, M. Sprossig, and H. Schweizer, Pharmazie 1981, 36(1), 50-53; K. D. Thiel, B. Helbig, M. Sprossig, R. Klocking, and P. Wutzler, Acta Virol. 1983, 27(3), 200-208; K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782; and G. Sydow, V. Wunderlich, R. Klocking, and B. Helbig, Pharmazie 1986, 41(12), 865-868.
A direct comparison of humic acids synthesized enzymatically and nonenzymatically from caffeic and hydrocaffeic acids has shown that the two synthetic routes produce materials that differ somewhat in their efficacy for the suppression of herpes (hominis) types 1 and 2 viruses: K. D. Thiel, P. Wutzler, B. Helbig, R. Klocking, M. Sprossig, and H. Schweizer, Pharmazie 1984, 39(11), 781-782.
German patent DE 3830333 C1 (Mar. 15, 1990) issued to Wagner discloses a pharmaceutical composition comprised in part of humic acid for the topical treatment of herpes virus-induced vesicular rash. The method of preparation of the humic acid utilized is not disclosed.
U.S. Pat. No. 4,999,202 (Mar. 12, 1991) issued to Cronje, et al disloses a composition that has bactericidal or bacteriostatic properties, and which comprises oxidized coal-derived humic acid or a salt or derivative thereof as the active ingredient in a suitable carrier. The active ingredient is preferably an alkali metal salt of coal-derived humic acid and the carrier is preferably water. The method of preparation involves recovery of the humic acid by precipitation, after acidification with an acid such as hydrochloric acid to a pH value of 2.
European patent application 0537430A1 (Apr. 21, 1993) from Riede, et al. discloses the use of natural or synthetic, modified or unmodified ammonium or alkali metal humates against viruses, especially against retroviruses such as HIV. Riede et al. disclose humates that have insignificant toxicity and are neither mutagens nor teratogens. Riede, et al. also disclose a specific synthetic preparation of said humates that requires as long as 10-15 days to complete the oxidation of the starting material during which time the reaction temperature is maintained below 40.degree. C. The solution is acidified to pH 4-5 following the synthesis, following which known methods of purification, such as preparative chromatography, ultrafiltration, centrifugation, or electrodialysis, are employed. No inorganic salts other than the oxidant or the starting material are employed during or after the synthesis.
World patent application 95/08335 (published Mar. 30, 1995) from Zanetti, which is equivalent to U.S. application Ser. No. 08/310,675 (filed Sep. 22, 1994) discloses a method of inhibiting human immunodeficiency virus infection that comprises contacting leukocytes, peripheral blood mononuclear cells, and lymphocytes of an individual infected with said virus with an anti-immunodeficiency virus amount of a natural, commercially available preparation of humic acid. Synthetic humic acid preparations are also disclosed. The synthetic procedure disclosed employs no inorganic salts other than sodium periodate for the oxidation of the starting material. The synthetic procedure employs acidification of the product of the synthesis with 6 M HCl to a pH of less than 1. This solution is allowed to stand overnight. A precipitate of the synthetic product forms which is washed several times with 1M HCl. The final step involves freeze drying the precipitate.
Phenolic polymers such as humic acid, when exposed to hydrochloric acid under the above conditions as well as the conditions in Cronje '202, may be chlorinated. That is, one or more chlorine atoms will possibly be added to the aromatic rings of the phenolic polymers: R. B. Wagner and H. D. Zook, Synthetic Organic Chemistry, New York: J. Wiley & Sons, March 1963, 88-147. Other changes such as selective O-demethylation of humic acid products may also occur in the presence of hydrochloric acid: M. Fieser and L. F. Fieser, Reagents For Organic Synthesis, New York, Wiley-Interscience, Vol. 4, 1974, 250. It has been reported that aqueous chlorination of humic acids results in the formation of compounds with direct-acting mutagenic activity in the Ames/Salmonella plate assay. Nonchlorinated humic acids are not mutagenic: J. R. Meier, R. D. Lingg, R. J. Bull, Mutat. Res., 1983, 118(1-2), 25-41. It has also been reported that freeze-dried, chlorinated humic acid contains nonvolatile, direct-acting mutagenic and/or alkylating agents: S. C. Agarwal, J. Neton, Sci. Total Environ., 1989, 79(1), 69-83. A subchronic 90-day toxicology study has been conducted with chlorinated and nonchlorinated humic acids using male Sprague-Dawley rats. Increased incidence and severity of hematuria was found in the 1.0-g/l chlorinated humic acid group: L. W. Condie, R. D. Laurie, J. P. Bercz, J. Toxicol. Environ. Health, 1985, 15(2), 305-14. Thus, synthetic methods for the production of humic acids that can possibly produce chlorinated humic acids are to be avoided.
Another area of related art relevant to this invention is comprised of blood product compositions and methods for treating blood products to reduce viral and microbial activity. A variety of human blood products including blood platelets exist to meet critical medical therapeutic needs. Viral safety depends upon donor selection and screening. It has proven to be impossible to date to screen blood products adequately to provide complete assurance that there is no viral contamination. These blood products may be inadvertantly contaminated with viruses such as HIV-human immunodeficiency virus, hepatitus virus, including hepatitus A, B, and C and other viruses. A solvent/detergent (SD) technique exists for treating blood products including blood platelets, but this technique is primarily limited to lipid enveloped viruses and is known to be ineffective for nonenveloped viruses such as hepatitus A, parvovirus B19 and picornaviruses: P. M. Mannucci, et al., Ann. Intern. Med., 1994, 120(1),1-7; and L. Gurtler, Infusionsther. Transfusionsmed., 1994, 21(Suppl 1), 77-9. Additionally, it is necessary to separate the detergents in the SD method from the blood product utilizing extraction with soybean or castor oil and chromatography on insolubilized C18 resin: B. Horowitz et al., Blood, 1992, 79(3), 826-31; and Y. Piquet et al., Vox Sang., 1992, 63(4), 251-6.
A pasteurization process has been developed for treating blood products. This involves heat treatment of a stabilized aqueous protein solution at 60.degree. C. for 10 hours. However, residual infectious hepatitus A virus has been found even after 10 hour heat treatment of the stabilized preparation: J. Hilfenhaus and T. Nowak, Vox Sang., 1994, 67(Suppl 1), 62-6. Neither the solvent/detergent (S/D) process nor the pasteurization process alone are adequate to inactivate viruses that are strongly resistant to heat and organic solvents. In this context, human parvovirus B19 and hepatitus A virus are of particular concern: H. Schwinn et al., Arznneimittelforschung, 1994, 44(2), 188-91.
A final super heat treatment (100.degree. C. for 30 min) has been developed as an additional virus inactivation step to improve the safety of plasma derived factor VIII (FVIII) concentrate already treated with the solvent/detergent (S/D) method during the manufacturing process. The efficiency of the super heat treatment was demonstrated in inactivating two nonlipid enveloped viruses (Hepatitus A virus and Poliovirus 1). However, the loss of FVIII procoagulant activity during the super heat treatment was about 15%, estimated both by clotting and chromogenic assays: S. Arrighi et al., Thromb. Haemost., 1995, 74(3), 863-73.
A method for treating human blood products employing short wavelength ultraviolet light (UVC) irradiation for virus inactivation and enhancement of its compatibility with proteins by quenchers of reactive oxygen species has been developed. However, blood protein recovery was typically only around 75%: S. Chin et al., Blood, 1995, 86(11), 4331-6. Ultraviolet irradiation methods have additionally been reported not to be applicable to cellular blood products: C. M. Allen, Photochem. Photobiol., 1995, 62(1), 184-9.
In summary, there remains a need for a safe, efficacious and simple method for treating all human blood products to reduce or eliminate lipid enveloped and nonenveloped virus activity without loss of blood product or blood product activity.
The diversity of physicochemical characteristics as well as wide variation in the biological activity and toxicity of humics extracted or otherwise derived from natural soils has been well documented. This diversity and variation is due to variations in factors such as the source of the soil, the method(s) of extraction and/or isolation, and the technique(s) employed to treat the extract once it has been separated and isolated from crude soil. The consequence of irreproducibility of the properties of substances extracted from natural soil is that the commercial value of such materials is minimized. In addition, they are rendered unsuitable as medicaments. Also, while a number of laboratory-scale processes have already been described that address various aspects of the isolation, synthesis, and/or preparation of humic substances or similar materials, there are no reports of preparing and isolating such purely synthetic humic acids or similar materials by methods that are suitable for scaleup directly to industrial levels, that provide economically acceptable yields, and that optimize the preparation procedures from the standpoint of medicament safety and efficacy. All of the known synthetic methods utilize potentially toxic precipitation methods (lead(II) nitrate precipitation) followed by complex isolation procedures, potentially mutagenic compound-producing hydrochloric acid precipitation or lengthy synthetic steps as long as 10 days. The solution is to devise simple synthetic procedures that yield inexpensive, safe materials whose physicochemical attributes are reproducible, and that at least simulate those of typical commercially-available soil extracts. This invention is directed to this solution and to compositions and methods employing synthetic materials prepared according to the process of the invention.